Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd Assembly of iron-bound ovotransferrin amyloid fibrils Zihao Wei a , Qingrong Huang a,b,* a Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, United States b College of Chemistry and Environmental Engineering, Wuyi University, Jiangmen, Guangdong 529020, China ARTICLE INFO Keywords: Self-assembly Ovotransferrin amyloid fibrils Atomic force microscopy β-sheet structure Periodicity In vitro cytotoxicity ABSTRACT The impacts of pH, temperature, ionic strength and stirring speed on the assembly of ovotransferrin (OVT) into amyloid fibrils were analyzed by using thioflavin T fluorescence and atomic force microscopy. Optimum OVT nanofibrillation condition was achieved at pH 2, 90 °C, an ionic strength of 150 mM and a stirring speed of 300 rpm. Apart from rigid and long amyloid fibrils, flexible and short amyloid fibrils were also detected under the optimal condition. Morphological changes observed by atomic force microscopy as a function of time de- monstrated that short OVT amyloid fibrils (with contour length below 800 nm) were generated upon heating for 1 h, and long OVT amyloid fibrils (with contour length above 800 nm) appeared after 6 h heating. In terms of structural characteristics, circular dichroism study revealed that internal structures of OVT amyloid fibrils could be stacked β-sheet. Analysis of fibril periodicity indicated that OVT amyloid fibrils might consist of 2 or 4 multi- stranded filaments. With the aid of ANS (1-anilino-8-naphthalensulfonate) fluorescence probe, it was found that OVT amyloid fibrils had lower surface hydrophobicity than untreated OVT. MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay showed that OVT amyloid fibrils had no in vitro cytotoxicity, implying great application potential in food. This work will advance our understanding of amyloid fibrils derived from iron-bound proteins. 1. Introduction Proteins may self-assemble in vitro into aggregates with different morphologies, and protein aggregates are amorphous in most cases (Wang, 2005). Formation of aggregates is often regarded as an indica- tion of protein instability and various means have been developed to inhibit aggregation (Wang, 2005). However, protein aggregation is desirable under certain circumstances (Gao et al., 2017; Peng, Simon, Venema, & van der Linden, 2016). Unlike random or spherical irre- versible/reversible aggregates, protein fibrils are anisotropic aggregates with linear structures (Jones & Mezzenga, 2012). Nanofibrils refer to fibrils with diameters less than 100 nm (Chinga-Carrasco, 2011), and most of protein fibrils can be considered as nanofibrils based on the definition (Mohammadian & Madadlou, 2018). Protein nanofibrils generated through self-assembly are also called protein amyloid fibrils (Hu et al., 2018). The unique rod-like structure and high aspect ratio endow protein amyloid fibrils with particular features such as unique interfacial adsorption behavior, rheological behavior or gelling beha- vior, thus making protein amyloid fibrils interesting and competitive candidates in application of Pickering emulsions, hydrogels, foam sta- bilization, nutraceutical delivery and so on (Gao et al., 2017; Hu et al., 2018; Li, Adamcik, & Mezzenga, 2012; Peng et al., 2016; Shen et al., 2017; Wan, Yang, & Sagis, 2016). Like most of potential new in- gredients and soft materials in food formulations, formation and structure of protein fibrils should be tailored to satisfy diverse needs in complex food systems (Mezzenga, Schurtenberger, Burbidge, & Michel, 2005). Therefore, a deeper understanding of fibrils is in urgent need to gain precise control over engineering finely structured fibrils. Ovotransferrin (OVT) is an iron-binding egg white protein con- taining 686 amino acids (Wu & Acero-Lopez, 2012). OVT is composed of two globular lobes (N and C lobes), and each lobe can bind one Fe 3+ ion with bicarbonate anions reversibly. The iron-binding ligands in each lobe of OVT contain one aspartic acid, one histidine and two tyrosine residues, and this set facilitates stronger iron binding (Wu & Acero-Lopez, 2012). OVT possesses a variety of bioactivities such as antimicrobial activity, anticancer activity and immune-modulating ac- tivity (Ibrahim & Kiyono, 2009; Kobayashi et al., 2015; Varon, Allen, Bennett, Mesak, & Scaman, 2013). Despite multifunctional properties of OVT, there has been little work done about OVT fibrils to our best knowledge. Till now, the published protein fibril studies have covered many globular proteins such as whey protein isolate (WPI), β-lacto- globulin (BLG), lysozyme, soybean proteins, bovine serum albumin and so on (Dave et al., 2013; Humblet-Hua, Sagis, & van der Linden, 2008; Loveday & Gunning, 2018; Mantovani, de Figueiredo Furtado, Netto, & https://doi.org/10.1016/j.foodhyd.2018.11.028 Received 2 October 2018; Received in revised form 12 November 2018; Accepted 12 November 2018 * Corresponding author. Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, United States. E-mail address: qhuang@sebs.rutgers.edu (Q. Huang). Food Hydrocolloids 89 (2019) 579–589 Available online 13 November 2018 0268-005X/ © 2018 Elsevier Ltd. All rights reserved. T